Clusters of genomic damage induced by therapeutic and environmental ionizing radiation (IR)/radiomimetic drugs include double-strand breaks (DSBs) with nonligatable ends, and more abundant clusters of base/sugar oxidation products, abasic (AP) sites and single-strand breaks (SSBs). Highly cytotoxic DSBs, also formed during SSB replication, activate cell-cycle checkpoints and promote DSB repair (DSBR), which in mammalian genomes occurs via nonhomologous end joining (NHEJ) in all cells, and by error-free homologous recombination (HR) in S/G2 cells. Alternative end joining (Alt-EJ) also repairs DSBs, including those generated during the repair of bi-stranded non-DSB lesion clusters by DNA glycosylases (DGs), AP-endonuclease (APE1), and others via the BER/SSBR pathway. NHEJ-initiating Ku inhibits Alt-EJ which using microhomology- based SSBR is more error prone than NHEJ. Radiosensitivity caused by DG/APE1 deficiency and of HR negative tumors by inhibition of PARP-1-initiated SSBR indicates Alt-EJ/SSBR's significant contribution to radioresistance, which must be coordinated with NHEJ, the predominant DSBR pathway in mammals.BER prior to NHEJ would cause secondary DSBs which near preexisting DSBs would lead to larger deletions. This project's central hypothesis is that NHEJ precedes Alt-EJ/BER, coordinated by Ku (Ku70/80), which recruits DNA-PKcs at the DSB, followed by the DSB's end processing and re-ligation by DNA ligase4/XRCC4/XLF. Based on our preliminary studies showing that: (a) Ku present in DG/APE1 immunocomplexes (ICs) inhibits them; (b) the Ku IC from irradiated cells performs NHEJ of a novel linearized plasmid substrate with dirty ends, whose in-cell repair involves both NHEJ and Alt-EJ, we hypothesize that hnRNP-U, present in Ku IC only after irradiation, and phosphorylated by DNA-PK during NHEJ, relieves Ku inhibition, thus acting as a molecular switch for transition to Alt-EJ/BER which utilizes SSBR proteins and a distinct set of end-processing enzymes. We will test various facets of this comprehensive hypothesis by pursuing three aims: Aim 1. To assess the contribution of Alt-EJ to radioresistance and its requirements in repairing radiation damage using reporter plasmid assays in-cell and in vitro, in parallel with analysis of cell genome repair, and to show that Alt-EJ/BER is additive to NHEJ in radioprotection. Aim 2. To test the hypothesis that hnRNP-U and Ku together coordinate NHEJ and Alt-EJ via DNA-PK-mediated phosphorylation. Aim 3. To test the hypothesis that NHEJ and Alt-EJ/BER proteins form repair-competent, dynamic complexes modulated by radiation/enediyne drugs involving physical interaction with Ku. We will characterize the end- processing enzymes and gap-filling DNA polymerase(s) for Alt-EJ. These studies will profoundly enhance our understanding of the repair of complex radiation-induced genomic damage, and of the contribution of Alt-EJ to radioresistance of tumors, and help identify novel therapeutic targets such as Ku for simultaneous radiosensitization of tumors and radioprotection of normal cells.